Micromanipulation and storage apparatus and methods

ABSTRACT

An apparatus for micromanipulation of biological material, includes a vessel (1) having a reservoir (2) wherein the vessel has a channel (3) formed in a portion of the reservoir, the channel including an intermediate restriction (4) dimensioned to resist passage of the biological material but allow passage of liquid treatment solutions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. Provisional PatentApplication No. 61/349,296 the content of which is incorporated hereinby reference.

INTRODUCTION TO THE INVENTION

This invention relates to apparatus and methods for themicromanipulation of biological materials and in particular, apparatusand methodologies for use in the cryopreservation of biologicalmaterials including human and non-human oocytes, embryos, sperm, stemcells and blastocyst.

Whilst the invention has been developed and has application in a widerange of micromanipulation situations and techniques with a range ofbiological materials, it finds particular application for use in thecryopreservation of human oocytes, embryos, morula, sperm and stem cellsby vitrification as applied during In Vitro Fertilisation (IVF)procedures and the like.

BACKGROUND OF THE INVENTION

The technologies involved and applied for cryopreserving of human andanimal embryos are well established and with the application of suitableskill and know-how, the current technologies have achieved greatimprovement in the reliability and success in In Vitro Fertilisationprocedures.

For the purposes of this specification, the term “freezing” and“vitrification” are taken to have the following definition:

“Freezing” is the cooling of a liquid to a solid state which may includecrystalisation.

“Vitrification” is the cooling of a liquid to a solid state withoutcrystalisation.

The techniques as understood and applied involve harvesting andcryopreservation of embryos, with a plurality of steps involvingharvesting and extraction of oocytes, in vitro fertilisation thereof andthe subsequent freezing and storing of such fertilised eggs and theresultant embryos and/or late stage blastocysts. The multitude of stepsand handling stages required are heavily reliant on a high level ofknow-how and skill via the technical operators. The embryos orblastocysts once frozen, are then made available as required and can bethawed and transferred to the recipient whereby successful implantationto the uterus can result in normal development of a fetus and aresultant pregnancy.

More recently, such cryopreservation techniques have been successfullyapplied to unfertilised eggs and oocytes. Oocyte cryopreservationinvolves harvesting, freezing and storing of eggs or oocytes from adonor female in an unfertilised state. Such frozen eggs can then bedrawn from a storage bank, thawed and made available for fertilisationand transferred to a donor on demand.

The techniques of cryopreservation as applied to oocytes rather thanfertilised eggs and embryos, has certain ethical and medical advantagesand has been subject to increased research and experimentation toimprove the techniques involved.

The process of cryopreservation, particularly when applied to “live”biological materials, involves a high degree of trauma for thebiological material in question, particularly having regard to themultiple handling steps required in accordance with current techniques.In addition to the trauma experienced as a result of physical handling,the biological material is also subject to potential ice crystalformation during freezing process, in addition to osmotic shock andtoxic shock experienced during movement through a plurality ofprocessing chemical solutions.

The traditional method of preparing frozen biological material includesthe slow cooling of the material and its surrounding solution down tothe storage temperature, with a view to deliberately initiating theformation of ice crystals remotely from the biological material per se.The traditional method is not optimal due to continuous formation of icecrystals. Alternative “vitrification” methods have been developed toaddress the ice crystal formation issues, however vitrification requiresconsiderable technical skill for successful execution. Vitrificationinvolves the transformation of the processing solution into a glass-likeamorphous solid that is free from any crystalline structure, followed byextremely rapid cooling. The extremely rapid cooling is what enables thesolution to achieve the glass-like amorphous state.

The application of either the traditional method of freezing orvitrification involves the use of chemical compounds and solutions,which are added to the biological material to minimise cell damageduring the freezing processes. The chemical compounds and solutions areknown as cryoprotectants and include permeating and non-permeatingsolutions. Permeating cryoprotectants are small molecules that readilypermeate the membranes of the biological material with the formation ofhydrogen bonds to the water molecules of the biological material withthe aim of preventing ice crystallisation thereof. Examples of suchpermeating cryoprotectants are Ethylene Glycol (EG), Dimethyl Sulphoxide(DMSO) and Glycerol. At low concentrations in water, such permeatingcryoprotectants lower the freezing temperature of the resultant solutionand can assist in the prevention and minimisation of icecrystallisation. At higher concentrations which may differ at differentcooling rates, such permeating cryoprotectants inhibit the formation oftypical ice crystals and can lead to the development of a solidglass-like or vitrified state in which water is solidified prior tocrystallisation or expansion. Toxicity of such permeatingcryoprotectants increases with their increasing concentrations and ispotentially toxic to the biological material in question andaccordingly, the biological material must have minimal exposure to thepermeating cryoprotectants over a very short period of time, oralternatively, exposure at a low temperature, whereby the metabolic rateof the biological material in question is reduced.

In contrast to the permeating cryoprotectants, the non-permeatingcryoprotectants remain extracellular. Some examples of non-permeatingcryoprotectants include disaccharides, trehalose and sucrose. Thedisaccharide cryoprotectants act by drawing free water from within thebiological material and dehydrating the intracellular spaces. Theresultant dehydration allows them to be used in combination withpermeating cryoprotectants, such that the net concentration of thepermeating cryoprotectant can be increased in the intracellular space.These techniques further assist the permeating cryoprotectant inpreventing or minimising ice crystal formation.

During the vitrification process, permeating cryoprotectants may beadded at a high concentration while the biological material'stemperature is controlled at a predetermined level above freezing.However, because the toxicity of such high concentrations of permeatingcryoprotectant can be substantial, it is not possible to retain thebiological material at such temperatures for extended periods.Alternatively, a reduced time can be allowed for equilibrium after whichthe biological material, which may include oocytes or embryos areplunged directly into liquid nitrogen to effect freezing. The extremelyrapid rate of cooling, minimises the negative effects of thecryoprotectant on the biological material and also, minimises icecrystal formation by encouraging vitrification.

The vitrification process involves exposing the biological material toat least three vitrification solutions. The vitrification solutions aretypically added to successive wells in a multi-well culture dish, wherethe dish and solutions are warmed to a predetermined temperature,determined in accordance with the requirements of the biologicalmaterial in question.

In a typical protocol, the biological material is physically transferredto a first solution in a first well and then washed by physically movingthe biological material or cell through the solution in question with acell pipetting device. The washing process is repeated in a second,third and fourth well over predetermined periods of time until thebiological material or cell is considered ready for cryopreservation.The biological material is then physically drawn up with a predeterminedamount of vitrification solution using a pipette or other handlingdevice. A droplet containing the biological material or cell to bevitrified is then pipetted onto the vitrification device. Thevitrification device is then physically transferred with the droplet andbiological material attached and directly plunged into liquid nitrogenor placed onto the surface of a vitrification block that has beenpre-cooled with liquid nitrogen. Once the biological material and thecarrying fluid have become vitrified, the vitrification device is theninserted into a pre-chilled straw or other storage device, located in aslot in the vitrification block for subsequent transfer to long-termcold storage in either liquid nitrogen or liquid nitrogen vapour.

Various vitrification devices are used to manipulate the sample duringthe cryopreservation processes. Some propose a pipette style device inwhich the sample is sucked into a hollow tube which is then plungeddirectly into the solution or liquid nitrogen. Such device is marketedby Irvine scientific and sold as Cryotip®.

Other uses a loop/hook style device which will have a close loop or anopen hook made from plastic or metal wire stuck to the end of a stem andis used to pick up the biological sample. Such devices are marketed byCryologic under the trade name of fibreplug or Cryoloop as defined inWO00/21365.

Others tools as disclosed in international application WO 02/085110“Cryotop” which is a flexible strip attached to a piece of plastic, inwhich the sample is placed on the strip and plunged directly into liquidnitrogen.

Current prior art requires many embryo handling steps using multipleapparatus; every handling step increases the chance of losing theembryo. It is estimated that 1-2% of embryos lost are contributed byhandling during the vitrification step.

The trauma associated with the previously described processes and inparticular the trauma imposed by repeated physical handing andmanipulation of extremely delicate biological material including eggs,cells, embryos and blastocysts, impacts on the survival rate and hencethe success, of the processes and methods previously described.Furthermore, the physical dynamics of a living embryo introduce rapidgrowing and changes to the shape of the embryo which further challengeany handling, and in particular, automated handling of such biologicalmaterials. Any automation needs to manage such dynamics as well asmanage a range of different embryo types, rapid fluid movement alongwith a high range of fluid viscosities. Clearly, in order to maximisethe chances of success and minimise trauma imposed on the materialsbeing handled, it is highly desirable to reduce the physical handling ofsuch delicate materials to an absolute minimum, in addition tominimising the number and degree of different washing solutions appliedto the biological material.

Culturing is a technique to grow embryos to day 4, 5 or 6 postfertilisation to assist in the selection of the best quality embryos fortransfer. Extended culture can increase the probability of a successfulpregnancy.

One object of the invention is to provide improved apparatus and methodsfor the micromanipulation and storage of biological materials includingbut not limited to the culturing and cryopreservation of such materials.

Another object of the invention is to control the washing protocol timesusing automation and to reduce handling of the embryo, thus enablingfull automation.

SUMMARY OF THE INVENTION Detailed Description

In a first aspect the invention provides an apparatus formicromanipulation of one or more biological materials, said apparatusincluding a vessel having a reservoir wherein said vessel has a channelformed in a portion of said reservoir, said channel including anintermediate restriction dimensioned to resist passage of saidbiological material but allow passage of liquid treatment solutions. Therestriction can include a generally vertical passage or a generallyhorizontal platform. The intermediate restriction may include a physicalnarrowing of the passage, an orifice partially blocking the channel orone or more divots or other partial blocking means. The liquid treatmentsolution can include culture medium, cryopreservation medium, thawingmedium, vitrification medium, fertilisation medium, or buffer solution.

The vessel preferably has an open top with the channel formed at thebottom thereof. The channel most preferably includes sub-reservoirs ateither end of the restriction. The vessel is preferably formed of amaterial with high thermal conductivity and diffusivity with aparticular preference for high conductivity in the sub-reservoir andchannel regions. The vessel preferably includes a cap or lid adapted toseal the open top as either a separate lid or an integral hinged lid.

The apparatus may include a holder for said vessel.

The vessel or holder preferably includes a portion adapted to receive abar code or other indicia.

The vessel material is preferably chosen from a material capable, ofbeing sterilised and is biologically inert so as to pose no threat ofcontamination to the biological material/s. The vessel materialpreferably has high thermal diffusion and most preferably is between 1mm and 0.05 mm thick.

In another aspect the invention provides an apparatus being an automatedcryopreservation device including provision for one or a plurality ofthe vessels as previously described wherein the apparatus furtherincludes means to irrigate the captive biological material withvitrification and/or other treatment solutions and means to vitrify thecaptive biological material.

In another aspect the invention provides a semi automated deviceincluding provision for one or a plurality of the vessels as previouslydescribed wherein the apparatus includes a means to irrigate the captivebiological material with pre-vitrification or vitrification treatmentsolutions and fluidics to prepare the captive biological material forvitrification.

In another aspect the invention provides a method of cryopreservation ofbiological material comprising the steps of:

-   -   introducing the biological material into one sub-reservoir        region of the previously described vessel irrigating and        draining the biological material with a series of vitrification        solutions introduced and removed from the other sub-reservoir        (at the other end of the channel from where the embryo is        introduced)    -   final draining of said irrigation solutions from the vessel    -   vitrifying the vessel and captive biological material.        In another aspect the invention provides a method of        cryopreservation of biological material comprising the steps of:    -   introducing the biological material into one sub-reservoir        region of the previously described vessel irrigating and        draining the biological material with a series of vitrification        solutions introduced and removed from the other sub-reservoir        (at the other end of the channel from where the embryo is        introduced)    -   dispensing minimal final vitrification solution    -   vitrifying the vessel and captive biological material.

The series of vitrification solutions preferably includes graduallyincreasing non-permeating and permeating cryoprotectants selected fromEthylene Glycol (EG), Dimethyl Sulphoxide (DMSO), Glycerol,disaccharides, trehalose and sucrose to enhance the sinking of saidbiological material to the bottom of said sub-reservoir region so as toallow maximum drainage and retention of minimal irrigation solutionprior to vitrification or freezing.

In another aspect the invention provides a method for pre-vitrificationtreatment of a biological material said method comprising the steps of:

-   -   introducing said biological material into the reservoir region        of an apparatus as previously described for capture in the        channel region of said vessel;    -   irrigating and draining said biological material with culturing        or pre-vitrification solutions and fluidics.

In another aspect the invention provides a method of thawing a vitrifiedbiological material and method comprising the steps of:

-   -   withdrawing a vitrified biological material in an apparatus as        previously described from cooling solution;    -   thawing the vessel and captive biological material by heating in        a heated solution.    -   heat can also be applied to the surface of the vessel or        surrounding air;    -   irrigating and draining said captive biological material with a        series of thawing solutions introduced and removed from the        reservoir region of said vessel;    -   draining said irrigation solutions;    -   recovering thawed biological material from said vessel.

In another aspect the invention provides a method of storing acryopreservation biological material comprising the steps of:

-   -   introducing said biological material into the reservoir region        of an apparatus as previously described for capture in the        channel region or said vessel;    -   irrigating and draining said biological material with a series        of vitrification solutions introduced and removed from the other        sub-reservoir (at the other end of the channel from where the        embryo is introduced);    -   final draining said irrigation solutions from said vessel;    -   vitrifying said vessel and captive biological material;    -   storing said vessel and captive biological material in a storage        facility until the biological material is devitrified.

In another aspect the invention provides a method of thawing a frozen orvitrified biological sample comprising reversing the previouslydescribed method by using thawing solutions.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

LEGEND

-   1. Vessel-   2. Reservoir-   3. Channel-   4. Restriction-   5. Open top-   6. Bottom-   7. Sub-reservoirs-   8. Channel ends-   9. Cap or lid-   10. Indicia platform-   11. Support frame-   12. Cassette-   13. Hinge-   14. Divot-   15. Holder-   16. Loader

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of the vessel;

FIG. 2 shows a top view of the open vessel;

FIG. 3 shows a cross-sectional view of the vessel of FIG. 2, taken alongline 3-3;

FIG. 4 shows a cross-sectional view of the vessel of FIG. 2, taken alongline 4-4;

FIG. 5 shows a perspective view of the vessel with divots;

FIG. 6 shows a top view of the open vessel with divots;

FIG. 7 shows a cross-sectional view of the vessel with divots of FIG. 6,taken along line 7-7;

FIG. 8 shows a cross-sectional view of the vessel with divots of FIG. 7,taken along line 8-8;

FIG. 9 shows the vessel and a holder;

FIG. 10 shows an alternative holder and cap;

FIG. 11 shows the cap fitted to the vessel in the holder;

FIG. 12 shows the vessel holder with integral cap;

FIG. 13 shows the holder with cap closed;

FIG. 14 shows an alternative cap or lid;

FIG. 15 shows a cap or lid with a handle;

FIG. 16 shows a cap or lid with a peel-able opening;

FIG. 17 shows a holder and vessel cassette;

FIG. 18 shows a storage loader;

FIG. 19 shows an alternative storage loader;

FIG. 20 shows a stacked loader;

FIG. 21 shows a Dewar storage loader;

FIG. 22 shows a tank storage system.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described with reference to the Legend andFigures in the context of human in vitro fertilisation and the handlingof biological material, being a fertilised human embryo, but the scopeof the invention extends broadly to a wide range of micromanipulationtechniques for biological material where the biological material inquestion can be conveniently handled and managed using the apparatus ofthe invention and made subject to a number of processing steps andprocedures without being physically moved from the confines and securityprovided for by the apparatus of the invention.

In its broadest aspect, the invention provides a convenient holdingvessel for receiving and securely holding a human embryo or other sampleof biological material. The vessel 1 is formed as a unitary hollowbodied vessel having an internal reservoir 2. The vessel is preferablydimensioned to a small scale with the reservoir occupying the bulk ofthe vessel. The preferred small size of the vessel is particularlyadvantageous by allowing the miniaturisation and minimalisation oftreatment solutions required for processing and vitrification of thebiological material. The preferred volume for the treatment solutions isbetween 0.1 ul-100 ul, more preferably 0.5 ul-10 ul. The vesselpreferably has an open top 5, giving direct access to the reservoir 2with the open top providing ready access for introduction of the embryoor other biological material and also easy access for introduction andflushing of the treatment solutions being used.

The vessel is preferably generally oval in shape and includes a channelportion 3 formed in the bottom region 6 thereof. The channel regionpreferably includes dual raised portions of the bottom region 6 of thevessel which extend longitudinally and include a restriction 4 formedtoward the centre thereof. In this manner, the integrally formed channelprovides a preliminary flow path for introduced treatment solutionswhich can be introduced into the reservoir and flushed along the channelfrom one end 8 to the other end 8. As more treatment solution isintroduced into the reservoir the flew path moves over the channel intothe bulk of the reservoir. The provision of the channel creates twosub-reservoirs 7 at either end of the vessel, communicating with andbeing formed integrally to the channel. The channel has a restriction 4at or near the middle or centre thereof, which is dimensioned to resistpassage of a human embryo or resist passage of whatever biologicalmaterial intended for use. The restriction is most preferablydimensioned to resist, but not totally prohibit passage of a humanembryo, but dimensioned to encourage the human embryo to remain withinone of the sub-reservoirs where the action of surface tension and fluiddynamics encourages the embryo to remain at one or other of thesub-reservoirs, rather than moving from one sub-reservoir to the otheras treatment liquids are flushed into the reservoir, and through thechannel.

As fluid is drawn through the channel away from the sub-reservoircontaining the embryo, the dimensions and cross-section of the channelrestrict fluid movement in a particular region in the manner of a “deadpocket”. This region is on the base of vessel, in the vicinity of thegradual transition of wider sub-reservoir to narrower channel. Thisconfiguration allows the captive embryo to be retained at one end andminimises treatment solution needed to flush the embryo.

The reservoir is preferably symmetric—when the fluid withdrawal positionis further away from the centreline of the channel than the restrictedflow region described above (i.e.—actually in the sub-reservoir), thesurface tension retaining fluid in the channel overcomes the surfacetension joining the fluid to the withdrawal position as it is furtheraway. The fluid eventually ‘breaks’, leaving a residual volume in thechannel, containing the embryo. The residual volume is sufficientlysmall for rapid vitrification of the embryo.

The geometry of the holding vessel contributes to the flowcharacteristics of fluid movement and contribute toward control andmanipulation of the embryo. For example, withdrawal of fluid too closeto the channel, will risk draining the channel totally and loosing theembryo. Conversely, if fluid is withdrawn too far away from the channel,too much fluid can be left behind which can compromise rapidvitrification. Moreover, the rate of fluid withdrawal is important toensure there is a “dead pocket”.

The floor of the sub-reservoirs preferably slope towards the channel toenable gravity to assist locating the embryo in the “dead pocket”. Theslope should preferably not continue into the channel, but mostpreferably has a flatter base to prevent the embryo from entering thechannel proper.

Referring to FIGS. 5 to 8, in a particularly preferred embodiment the“dead pocket” can be provided by way of the provision of one of moredivots 14 formed in the intermediate restriction 4, wherein the divots14 comprise one or more minor indentations, dimensioned to capture thebiological material or embryo within a small pocket within theintermediate restriction 4. In this manner, the divots 14 provide anumber of advantages including the provision of a fixed and specificlocation for the end user to position the biological material insertedinto the apparatus of the invention. The divot 14 provides a specificlocation and helps ensure the biological material ends up beingpositioned in a highly specific location during the operation of fluidexchange. The divot 14 further provides a barrier to prevent the embryoor biological material from being aspirated away. The provision of twodivots allows asymmetrical positioning where a first divot is positionedthe start of the restriction and a second divot positioned at the middleof the restriction.

In addition, the provision of a divot 14 allows for maximum removal ofprevious irrigation solutions and in particular, the divot 14 allows ahighly controlled volume of leftover solution surrounding the capturedbiological material or embryo and therefore allows the reliabledispensation of a minimal amount of vitrification solution whilstknowing the precise position of the embryo of biological material withinthe apparatus.

Diameter of the withdrawal tip may contribute to the fluid bridgingbetween the sides of the sub-reservoir and assisting fluid removal andflow patterns.

In this manner, the vessel of the invention provides for the highlycontrolled handling and manipulation of human embryos and the like,where the human embryo can be carefully placed into the vessel at one orother of the sub-reservoirs 7 or directly into the divot. Once theembryo is carefully positioned in the sub-reservoir or divot, thetreatment fluids can be then carefully introduced into the vacantsub-reservoir which is not occupied by the biological material and withthe gentle flushing of fluid from an unoccupied or vacant sub-reservoirthrough the channel to the occupied sub-reservoir, the treatment fluidscan be withdrawn and the subsequent treatment fluids are then introducedinto the unoccupied sub-reservoir and gently flushed through to theoccupied sub-reservoir. In this manner, a plurality of flushingoperations can be executed with minimal disturbance of the embryo whichis held captive in the occupied sub-reservoir or divot using the vacantsub-reservoir to add or withdraw fluids.

In a particularly preferred embodiment, the treatment fluids can begraduated such that one continuous flushing operation can be executedusing a gradation of treatment fluids so as to minimise trauma and toxicshock to the embryo held captive in a first sub-reservoir. In this way,the embryo is subject to only one act of physical flushing, therebygreatly minimising any disturbance of the embryo. The configuration ofthe channel and restrictions is adapted to encourage flushing of fluidstherethrough, whilst minimising any unnecessary turbulence which couldpotentially buffet and physically damage the embryo held captive in thefirst sub-reservoir.

The treatment fluids preferably contain gradually increasingnon-permeating cryoprotectants and permeating cryoprotectants selectedfrom Ethylene Glycol (EG), Dimethyl Sulphoxide (DMSO), Glycerol,disaccharides, trehalose and sucrose which are specifically adapted toenhance or encourage the sinking of the embryo or biological material tothe bottom of the vessel. This enhances maximum drainage from the vesseland retention of minimal irrigation solution prior to vitrification orfreezing, thereby enhancing the vitrification or freezing step. Inaddition, by ensuring the biological sample the embryo rapidly sinks tothe bottom of the vessel, the chances of the biological material orembryo inadvertently escaping the vessel is minimised.

The vessel is preferably formed of a material having a high degree ofthermal diffusivity to ensure fast cooling rate. In addition, thematerial forming the vessel is preferably biologically inert to minimiseany possible contamination of the biological material. When thetreatment liquids have all been removed, the vessel is adapted forvitrifying by placement in liquid nitrogen or the like, such that thecaptive embryo is itself vitrified rapidly by the high thermalconductivity and diffusivity of the vessel materials. In this way, thepositioning of the embryo in the sub-reservoir at the bottom of thevessel, allows the vessel to be placed in liquid nitrogen, such that theliquid nitrogen has maximum thermal transmission directly to the embryo,whilst quarantining the embryo from any direct contact with the liquidnitrogen or other vitrification medium per se. Once the vitrificationprocess is accomplished, the vessel and captive (now vitrified) embryo,can be removed from the liquid nitrogen and the vessel stored in avitrified state awaiting thawing and use of the captive embryo, oralternative biological material.

An additional advantage of the vessel of the invention, particularly thevessel including the optional provision of divots and the minimalvitrification fluid used in the cooling process involves thawing, wherethawing of the frozen biological material within the vessel of theinvention can be accomplished rapidly with minimal disruption, due tothe minimal amount of solution surrounding the biological material asachieved by the provision of the small divot or divots. The staledvessel of the invention can be placed directly into a warm, solution toachieve rapid and controlled thawing whilst maintaining maximumviability of the biological sample.

The vessel may be provided with a dedicated holder 15 as shown in FIGS.9 to 13.

In order to maximise the versatility of the vessel and apparatus of theinvention, the vessel and/or holder are preferably provided with a capor lid 9, variations of the cap or lid being shown in FIGS. 14 to 16.The lid can be formed as a separate item or hinged directly and isadapted for snug fit to the open top 5 region of the vessel 1 and ismost preferably adapted to cooperate with the channel formation so as tosnap-fit and close the reservoir and provide minimal open space withinthe closed vessel. The provision of a lid assists in the handling andmanipulation of the vessel, particularly when it is placed into liquidnitrogen and subsequently vitrified and stored for future use. The lidalso protects the vitrified embryo from contamination and damage whilstin the vitrified state.

The lid is preferably formed integral to the vessel holder as shown inFIGS. 12 and 13 by way of an integral hinge 13 formed between the bodyof the vessel 1 or holder 15 and the lid 9. In this manner, the vesselis formed as a one-piece item thereby minimising the need to locate andprovide a separate seal for the vessel and provides a ready means ofsealing and closing the vessel immediately at hand.

Sealing can be either mechanical seal as describe above or chemical heatseal as shown in FIGS. 10 and 11.

The apparatus of the invention also includes a physical region thereoffor the provision of a bar code or other identifying indicia whereby theindicia platform 10 allows the ready and secure identification of thevitrified embryo or other item of biological material. The vesselthereby provides maximum security and identification for a given humanembryo, particularly as the biological material in question undergoeseffectively one handling step only during the whole process postharvesting, through to vitrification and thawing. In this manner, theembryo has little or no chance of being mislabelled or misidentifiedduring multiple steps and processes required for preparation andvitrification as the embryo never leaves the confines of the vessel oncethe treatment commences.

In another aspect the invention provides a cryopreservation apparatuswhere reference to FIGS. 17 to 21 details the adaptation of the vessel 1of the invention which can be provided with a cassette 12, adapted tohold a plurality of sealed vessels. The cassette is preferably designedto accept open vessels and to be used throughout the vitrificationprocess from start to end. Up to 4 vessels can be placed in the cassetteprior to processing, once the biological material is irrigated with thevitrification solution, the lid can be closed/heat sealed and plungedinto liquid nitrogen. In another aspect the cassette is designed toreceive sealed vessels once the embryos have been processed andvitrified and subsequently sealed into the closed vessels as previouslydescribed. A plurality of vessels in the cassette are subsequentlyplaced in a Canister which is then stored in a Dewar storage facility asdetailed in FIG. 21 or alternative storage. The cassette preferablyincludes a portion adapted to receive a bar code or other indicia 10.

The cassettes are adapted for placement in a loader 16 as shown in FIGS.18 to 20 with the current storage system is shown in FIG. 21.

An alternative storage is shown in FIG. 22, being a tank storageconcept.

The tank storage concept has advantages over current systems by:

-   -   Increasing capacity of samples being able to store    -   Easier retrieval by large identification areas located on both        the cassette and vessel    -   Partitioned to allow both cassettes and vessels to be located in        a fixed position

The apparatus and in particular the vessel component of the inventionlends itself to automation where the vessels can be automaticallyhandled by suitable automated apparatus such that the array of vesselsprovided in an automated example of the invention, can, once the embryoshave been carefully positioned into the vessel, then allow the fullautomation of the vitrification/freezing preparations andvitrification/freezing steps and the subsequent vitrifying/freezing andstorage of a plurality of embryos to be fully automated without therequirement for the embryo to be moved, or requirement for individualmonitoring or handling during any of the subsequent steps.

In another aspect the invention provides a semi automated deviceincluding provision for one or a plurality of the vessels as previouslydescribed wherein the apparatus includes a means to irrigate the captivebiological material with pre-freezing or vitrification treatmentsolutions and fluidics to prepare the captive biological material forvitrification.

The vessel of the invention also finds various other uses including:

-   -   irrigation said biological material with culturing media        (culturing);    -   irrigation said biological material with vitrification        solutions;    -   vitrifying said vessel and captive biological material;    -   storage;    -   thawing said vessel;    -   irrigation said biological material with thawing solution.

The full automation of the in vitro fertilisation preparation ofvitrified embryos is expected to provide substantial improvements toviability of the rendered embryos, thereby maximising the viability ofembryos once thawed and consequently maximising the chance of successand pregnancy to recipients of embryos provided by way of the apparatusand methods of the invention.

In addition to the apparatus the invention also provides a range ofmethods for the handling of biological materials as described.

In another aspect the invention provides a method of cryopreservation ofbiological materials comprising the steps of introducing a selectedbiological material into the reservoir region of a vessel as previouslydescribed for capture in the channel region thereof;

-   -   irrigating and draining said biological material with a series        of vitrification solutions introduced and removed from the other        sub-reservoir (at the other end of the channel from where the        embryo is introduced);    -   final draining said irrigation solutions from said vessel;    -   vitrifying said vessel and captive biological material;

In the most particularly preferred embodiment where the apparatusincludes the optional provision of one or a plurality of divotspositioned within the intermediate restriction a typical protocol wouldconsist of moving the embryo of biological material from a series ofvitrification media to the final and most active solution. The mediacomposition and time spent in each solution may be as follows:

Media Time spent in solution Solution 1: “Cryobase” 1-15 minutes Basicsolution Solution 2: “Vitrification solution 3-12 minutes 1” Media with7-10% of DMSO and EG Solution 3: “Vitrification solution 2” 30 sec to upto 3 minute Media with 15-18% DMSO and EG plus non permeatingcryoprotectants.

Listed below are examples of variations in protocol of cryopreservationof biological material using the described apparatus.

Protocol Example 1

Protocol of cryopreservation of biological material comprising of:

-   -   1. Introducing said biological material into the reservoir        region of the vessel with cryobase.    -   2. The cryobase is drained from the aspirate/dispense position.    -   3. Vitrification solution 1 is dispensed filling the entire        channel.    -   4. The biological material is then allowed to equilibrate in        vitrification solution 1.    -   5. Vitrification solution 1 is drained from the        aspirate/dispense position, leaving minimal amount of        vitrification solution 1.    -   6. Vitrification solution 2 is dispensed filling the entire        channel.    -   7. The biological material is then equilibrated for a very short        time.    -   8. Vitrification solution 2 is drained from the        aspirate/dispense position leaving minimal amount of        vitrification solution 2.    -   9. The vessel and the biological material are then vitrified in        liquid nitrogen.

Protocol Example 2

An alternative protocol is provided, without the removal ofvitrification solution 2 as follows:

-   -   1. Biological material is placed into the vessel directly into        the divot with cryobase.    -   2. The cryobase is drained from the aspirate/dispense position.    -   3. Vitrification solution 1 is dispensed into and filling the        entire channel.    -   4. The biological material is then allowed to equilibrate in        vitrification solution 1, the vessel is dimensioned so that the        biological material will roll back into the divot.    -   5. Vitrification solution 1 is drained from the        aspirate/dispense position, leaving minimal amount of        vitrification solution 1 in the divot.    -   6. Small volume of vitrification solution 2 (between 0.5 ul-2.5        ul) is dispensed into the channel covering both the divot and        biological material.    -   7. The biological material is then equilibrated for a very short        time.    -   8. The vessel and the biological material are then vitrified in        liquid nitrogen.

Protocol Example 3

In addition, a protocol involving the addition of non-permeatingcryoprotectant to the cryobase/vitrification solution 1, is provided asfollows:

-   -   1. Biological material is placed into the vessel directly into        the divot with cryobase.    -   2. The cryobase is drained from the aspirate/dispense position.    -   3. Vitrification solution 1 is dispensed into and filling the        entire channel.    -   4. The biological material is then allowed to equilibrate in        vitrification solution 1, the vessel is dimensioned so that the        biological material will roll back into the divot.    -   5. Vitrification solution 1 is drained from the        aspirate/dispense position, leaving minimal amount of        vitrification solution 1 in the divot.    -   6. Vitrification solution 2 is dispensed into the channel        covering the entire channel.    -   7. The biological material is then equilibrated for a very short        time.    -   8. Vitrification solution 2 is drained from the        aspirate/dispense position, leaving minimal amount of        vitrification solution 2 in the divot.    -   9. The vessel and the biological material are then vitrified in        liquid nitrogen.

Protocol Example 4

In addition, a protocol involving a single vitrification solutiongradually added, is provided as follows:

-   -   1. Biological material is placed into the vessel directly into        the divot with cryobase.    -   2. The cryobase is drained from the aspirate/dispense position.    -   3. Single vitrification solution is gradually introduced into        the sub-reservoirs and filling the entire channel.    -   4. The biological material is then allowed to equilibrate in        vitrification solution, ensuring the biological material sinks        to the bottom and remains in the divot or the intermediate        restriction.    -   5. Vitrification solution is drained from the aspirate/dispense        position, leaving minimal amount of vitrification solution.    -   6. The vessel and the biological material are then vitrified in        liquid nitrogen.

In another aspect the invention provides a method for pre-vitrificationtreatment of a biological material, said method comprising the steps of:

-   -   introducing said biological material into the reservoir region        of the apparatus as previously described for capture in the        channel region of said vessel;    -   irrigating and draining said biological material with culturing        for pre-vitrification solutions and fluidics.

In another aspect the invention provides a method of thawing a vitrifiedbiological material, said method comprising the steps of:

-   -   withdrawing a biological material vitrified in an apparatus as        previously described from cooling solution;    -   thawing the vessel of said apparatus and said biological        material by application of heat in a heated solution, applying        heat to the surface of the vessel or applying heat to the        surrounding area of said vessel;    -   irrigating and draining said captive biological material with a        series of thawing solutions introduced and removed from the        reservoir region of said vessel;    -   draining said irrigation solutions;    -   recovering thawed biological material from said vessel.

In a further aspect the invention provides a method of storing acryopreservation biological material comprising the steps of:

-   -   introducing said biological material into the reservoir region        of an apparatus as previously described for capture in the        channel region of said vessel;    -   irrigating and draining said biological material with a series        of vitrification solutions introduced into the first of said        sub-reservoirs and removed from the second of said        sub-reservoirs;    -   a final draining of said irrigation solutions from said vessel;    -   vitrifying said vessel and captive biological material;    -   storing said vessel and captive biological material in a storage        facility until the biological material is devitrified.

The methods of the invention lend themselves to a high degree ofautomation where the methods can be executed in automated apparatus anddevices such that a plurality of embryos or other biological materialcan be processed simultaneously. The particular features of theinvention allow for a high number of embryos or other biologicalmaterial samples to be manually introduced into the vessels of theinvention.

Once the individual samples of biological material have been introducedinto a dedicated vessel and the vessels installed in the automatedapparatus as previously described, the whole process of preparation forvitrification can be totally automated and monitored such that a largenumber of biological samples can be competently processed and vitrifiedin a seamless single-step operation.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the scope of theinvention as broadly described. The present embodiments are, therefore,to be considered in all respects as illustrative and not restrictive:

What is claimed is:
 1. An apparatus for micromanipulation of at leastone biological material, said apparatus including a vessel defining: areservoir with an open top; a channel formed at a bottom of thereservoir opposite the open top; a first sub-reservoir at a first end ofthe channel; and a second sub-reservoir at a second end of the channel,wherein the open top extends from the first sub-reservoir to the secondsub-reservoir, and wherein the channel narrows between the first andsecond sub-reservoirs to form an intermediate restriction dimensioned toresist passage of said biological material but allow passage of liquidtreatment solutions.
 2. An apparatus according to claim 1, wherein saidintermediate restriction is dimensioned to resist passage of an embryobut allow passage of liquid treatment solutions.
 3. An apparatusaccording to claim 1, wherein said vessel defines gradual transitionsbetween the intermediate restriction and the sub-reservoirs which arerelatively wider than the intermediate restriction.
 4. An apparatusaccording to claim 1, wherein a floor of each sub-reservoir slopes downtowards the channel at the bottom of the vessel.
 5. An apparatusaccording to claim 1, wherein said vessel is formed of a thermallydiffusive material.
 6. An apparatus according to claim 1, wherein saidvessel optimises heat transfer in the region of said sub-reservoirs andchannel.
 7. An apparatus according to claim 1, including one of a capand lid adapted to seal the open top of said vessel.
 8. An apparatusaccording to claim 1, including a portion adapted to receive at leastone of a bar-code and other indicia.
 9. An apparatus according to claim1, wherein the restriction is defined by convex surfaces of side wallsof the channel.
 10. An apparatus according to claim 9, wherein theconvex surfaces are curved in a first direction over at least part of alength of a channel, and curved in a second direction over at least partof a depth of the channel.
 11. An apparatus according to claim 1,wherein the vessel further defines one or more divots in the bottom ofthe vessel.
 12. An apparatus according to claim 11, wherein at least oneof the one or more divots is dimensioned to capture the biologicalmaterial within a small pocket within the intermediate restriction. 13.An apparatus according to claim 11, wherein the one or more divotscomprise a first divot positioned near a start of the restriction and asecond divot positioned at the middle of the restriction.
 14. Anapparatus according to claim 1, wherein a material of the vessel has athickness between 1 mm and 0.05 mm.